Interleukin-6 (IL-6) is a member of the helical cytokine family. IL-6 is produced by almost all cell types in response to a variety of different stimuli including bacterial (LPS) and viral infections, cancer, and other cytokines (e.g. IL-1). IL-6 is a pleiotropic factor, it participates in numerous processes and is thus associated with numerous disorders (for a review see Hirano T., Intern. Rev. Immunol. 16:249, 1998).
Bioactivity of IL-6 requires interaction of the cytokine, IL-6, its receptor (IL-6R) and a transmembrane signal transducer known as glycoprotein 130 (gp130), and formation of a hexameric complex containing two units of each protein. The outcome of the complex formation is dimerization of gp130, which by itself is sufficient for obtaining IL-6 like bioactivity (Fourcin, et al., J. Biol. Chem. 271: 11756, 1996.). Several other cytokines also use gp130 for signal transduction. These include: interleukin-11 (IL-11), ciliary neurotrophic factor (CNTF), leukocyte inhibitory factor (LIF), oncostatin M (OSM).
IL-6 and Host Defense
IL-6 levels increases early during bacterial and viral infections. IL-6 induces production of acute phase proteins which are thought to participate in the defense of the host organism against tissue damage and infection. The acute phase response is considered to be the systemic inflammatory reaction to infection and injury.
IL-6 also amplifies the immune system through its multiple growth and differentiation activities such as induction of B cell differentiation, replication of bone marrow progenitor cells, and augmentation of T lymphocytes, including enhancement of cytotoxic T lymphocytes.
IL-6 in Homeostasis, Injury and Transplantation
IL-6 levels are increased during stress. IL-6 in rabbits is directly responsible for elevation of body temperature. High IL-6 levels in burn patients correlates with mortality. Elevated IL-6 levels are associated with traumatic events and allograft rejection.
IL-6 in Osteoporosis
Estrogen plays an important role in maintaining bone mass. A massive loss of bone mass is reported in women at there postmenopausal stage. Increased bone resorption and increased osteoclast activity in postmenopausal osteoporosis have been linked with IL-6. The production of IL-6 is elevated in bone marrow cells at this stage correlating the fact that estrogen down regulates IL-6 gene expression. Estrogen loss induced by ovariectomy in mice enhances osteoclast development and this change can be prevented by antibodies to IL-6. Several experiments including an IL-6 knockout mice model, treatment with anti IL-6 antibodies or with IL-6 antisense demonstrate that elevated levels of IL-6 plays a critical role in the formation of osteoclastic cells. As such, dysregulation of IL-6 activity in bone cells leads to the development of pathological disease.
IL-6 in Immune Disorders
Elevated levels of IL-6 in cardiac myxoma and cervical carcinoma are associated with autoimmunity indications such as: production of anti-nuclear factor, rheumatoid factors, elevated immune complexes, arthritis and nephritis.
Castelman's disease patients suffer form fever, anemia, hyper-γ-globulinemia, and an increase in acute phase proteins.
The lymph nodes constitutively produce large amounts of IL-6, and surgical removal of the involved lymph nodes is followed by decrease in serum IL-6 levels and a dramatic clinical improvement. It has been demonstrated that systemic manifestation of Castelman's disease could be alleviated by treatment with anti-IL-6 antibody.
Local and general symptoms of rheumatoid arthritis, such as plasma cell infiltration into synovial tissues, autoantibody production, and polyclonal hyper-γ-globulinemia can be explained by increased IL-6 production observed in synovial tissue. At least two mediators which are elevated in RA patients, PGE2 and IL-1, are known to induce synthesis of IL-6. Higher than normal levels of IL-6 have been detected in sera of patients with active SLE. Increased plasma levels of IL-6 were observed in psoriasis patients.
AIDS
High levels of IL-6 are associated with HIV infection. The HIV envelope glycoproteins gp120 and gp160 induce IL-6 production from CD4+ T cells. Is has been demonstrated that IL-6 is a growth factor of the AIDS associated Kaposi's sarcoma (Murakamin-Mori, et al. Int. Immunol. 8: 595, 1996). The soluble form of the IL-6 receptor (sIL-6Ra) is a potent growth factor for AIDS-associated Kaposi's sarcoma (KS) cells. The soluble form of gp130 is antagonistic for sIL-6Ra-induced AIDS-KS cell growth. Furthermore, high IL-6 levels are associated with weight loss in AIDS.
Proliferative Diseases and Malignancies
An autocrine role for IL-6 has been reported in several types of cancer, among which are renal cell carcinoma, Hodgkin and non-Hodgkin's lymphoma, chronic lymphocytic leukemia, and acute myeloid leukemias. Plasmacytoma and myeloma cells require IL-6 for growth. Treatment of primary plasma cell leukemia with anti-IL-6 antibodies improves the patient's clinical status throughout the treatment. Also, IL-6 deficient mice are completely resistant to plasmacytoma induction.
Multiple myeloma is a malignant proliferation of plasma cells derived from a single clone. It is manifested in a number of organ dysfunctions and symptoms of bone pain or fractures, hypercalcemia, renal failure, susceptibility to infection, anemia and bleeding. The disease typically follows a chronic course for 2 to 5 years before progressing into an acute terminal phase.
The different therapeutic strategies for inhibition of multiple myeloma have been recently reviewed (Ogata A., Leuk. Res. 20 : 303, 1996). The vast majority of multiple myeloma patients require systemic chemotherapy to control the malignancy, and symptomatic supportive care to minimize the morbidity. The outcome of patients with multiple myeloma is still unsatisfactory, with median survival times of 2 to 3 years. Clearly, there is a need for an agent that cannot only improve the chances of remission, but also increase the duration of response and enhance survival.
Recently, injection of an anti-IL-6 antibody was tested in young population and resulted in a complete blocking of myeloma cell proliferation and inhibition of the serum IL-6 bioactivity. However, the administration of a single anti-IL-6 mAb appeared to be insufficient (Klein, et al., Blood 85: 863, 1995).
IL-6 has also a regulatory role in activation of Matrix Metalloproteinases (MMPs). MMPs are enzymes that are capable degrading the basement membrane components. As such MMPs are refereed as key enzymes in Extra Cellular Matrix remodeling, tumor invasion and metastasis.
Inhibitors of IL-6 Activity
It is generally accepted that IL-6 inhibitors have clinical value. As indicated above there are a number of clinical situations where IL-6 inhibitors could be of therapeutic use. Most of the attempts to produced inhibitors to IL-6 reported in the literature in the past, used proteins. In general, proteins are not very suitable as drugs, due to their immunogenic potential, high cost, and the necessity for parenteral administration. The various attempts to use proteins to inhibit IL-6 are described below.
Monoclonal Antibodies and Antibody Fragments
The most common approach is to use monoclonal antibodies (mAbs). Several murine mAbs capable of inhibiting the bioactivity of IL-6 have been described.
The drawbacks in the use of antibodies against IL-6 are that the mAb traps the IL-6 in an immune complex in the circulation (May et al., J. Immunol. 151, 3225, 1993), thereby increasing its half-life 200-fold (Lu et al., Blood 86, 3123,1995). The immune complexes are thus serve as long term, slow release deposits of IL-6. The presence of high levels of circulating immune complexes could result in their precipitation in the basal lamina in the kidneys or in the joints, which could lead to kidney failure or arthritis.
Attempts to block IL-6 with monoclonal antibodies have been reported for the following diseases: AIDS associated syndromes and lymphoma (Emilie, et al., Blood 84: 2472, 1994), Castelman's disease, rheumatoid arthritis (Wijdenes et al., J. Interferon Res. 14: 297, 1994, Yoshizaki et al. Springer Semin. Immunopathol., 20:247, 1998), multiple myeloma, plasma cell leukemia (Klein et al., Blood 78: 1198, 1991), and endotoxin toxicity. Partial response were observe in most instances, but the problems associated with the use of monoclonal antibodies for inhibition of IL-6 have so far prevented their routine clinical application.
Some of problems associated with the clinical use of monoclonal antibodies are a result of the large size of the antibody molecule. Minibodies which utilize the hypervariable loop structure of antibodies, capable of inhibiting IL-6 bioactivity were recently reported (Martin et al., The EMBO J. 13, 5303, 1994). Binding of IL-6 to a minibody molecule should create a complex that is small enough to be secreted from the kidney, thereby decreasing the risk of creating slow release IL-6 deposits. Minibody-IL-6 complexes may not be recognized as immune complexes, thereby decreasing the chances for kidneys and arthritic problems. The minibodies could still be immunogenic and it is unlikely that they will be orally available. So far, minibodies with sufficiently high affinity for IL-6 have not been obtained.
Mutated Proteins
Several IL-6 mutants were selected for desired activity using phage display systems. Super active mutants were reported (Toniatti, et al., The EMBO J. 15: 2726, 1996) as well as mutants which retain the capacity for binding of the IL-6R but lose the ability for interaction with the gp130 and thus could serve as functional antagonists of IL-6 bioactivity (Savino et al., The EMBO J. 13,5863, 1994; Sporeno, et al., Blood 87: 4510, 1996). The danger in clinical use of such mutants is the formation of antibodies that would recognize both the mutated and the native molecules. Such antibodies could block the bioactivity of IL-6 long after the treatment is terminated thereby exposing the patients to danger associated with lack of IL-6.
U.S. Pat. No. 5,470,952 disclose CTNF and IL-6 antagonists which are heterodimer proteins comprising a soluble α specificity determining cytokine receptor component and the extracellular domain of a β receptor component. Specifically, the inventors claim an IL-6 antagonist, capable of binding IL-6 to form a nonfunctional complex, comprising: soluble IL-6Rα and the extracellular domain of gp130.
Cytokine-toxin Conjugates
Several applications of IL-6 inhibitors entail the elimination of IL-6 dependent tumors, such as multiple myeloma. This goal can be achieved by the use of IL-6-toxin conjugate (Jean and Murphy, Prot. Eng. 4, 989, 1991). Malignant cells that have receptor for IL-6 would bind the toxin via the IL-6 portion of the conjugate and would be eliminated by toxin activity. Toxicity to all non-malignant cells that also express the IL-6 receptor is a dangerous possibility. Since IL-6 is required for development of normal humoral and cellular immune response, it is possible to speculate that treated patients would immunocompromised.
Peptide Antagonists of IL-6
Grube and Cochran identified a regulatory domain of the IL-6 receptor (J. Biol. Chem. 269: 20791, 1994). The region is from the extramembranal domain of the IL-6R and it is involved in the regulation of IL-6 signal transmission. A synthetic peptide, corresponding to residues 249-264 of the IL-6R inhibits IL-6-dependent cell mitogenesis and IL-6-stimulated acute phase response without affecting ligand binding.
In a search for possible lead compounds, epitope mapping of the human IL-6R was carried out (Halimi et al., Eur. Cytokin, Netw. 6:135, 1995) with mabs to IL-6R which inhibit the biological activity of IL-6 (Novick, et al., Hybridoma 10, 137, 1991). The 10 mer linear peptides that were recognized by two of the antibodies are from the same region identified by Grube and Cochran (ibid). The peptides identified by these two groups are indicated in the frame of the IL-6R sequence in FIG. 1 (* Grube and Cochran, ** Halimi et al.).
International PCT application WO 97/13781 discloses these synthetic peptides and analogs derived from the IL-6 that inhibit IL-6 activity. The peptides claimed are characterized also by being a linear epitope recognized by one or more Mabs specific to IL-6R. Peptides 1122 and 1123 (Halimi et al. ibid), were synthesized and found to inhibit IL-6 bioactivity in vitro with an ID50 of about 100 μM.
International PCT application WO 95/13086 and U.S. Pat. No. 5,420,109, discloses peptides which are cytokine restraining agents having the general formula: X1-X2-His-DPhe-Arg-DTrp-X3. These peptides are non-specific agents which modulate the activity of various cytokines (TNF, IL-1, IL-6 and IL-8) simultaneously, and therefore are not specific IL-6 inhibitors.
International PCT application WO 97/48728, discloses synthetic peptides which derived from IL-6 and from IL-6 receptor (either the IL-6R or gp130), and have IL-6 antagonistic or agonistic activity. The peptides interact with the receptor site of IL-6 or with IL-6Rs present at target cells or when combined, interact with both sites (IL-6 and IL-6R).
U.S. Pat. No. 5,210,075 discloses IL-6 antagonist peptides of varying length, which are modeled after a portion of the sequence of IL-6 itself (p51-70 Hirano et al. Nature 324:73, 1986), or which are modeled after four different portions of the sequence of the IL-6 receptor molecule.
None of these disclose conformationally constrained IL-6 peptide antagonists which are cyclized as described in the present invention.
Peptide Mimetic and Backbone Cyclized Peptide Analog Antagonists of IL-6
It is most beneficial to produce conformationally constrained peptide analogs overcoming the drawbacks of the native peptide molecules (low metabolic stability, poor oral bio-availability, rapid liver and kidney excretion, and lack of selectivity), thereby providing improved therapeutic properties.
A novel conceptual approach to the conformational constraint of peptides was introduced by Gilon, et al., (Biopolymers 31:745, 1991), who proposed backbone to backbone cyclization of peptides. The theoretical advantages of this strategy include the ability to effect cyclization via the carbons or nitrogens of the peptide backbone without interfering with side chains that may be crucial for interaction with the specific receptor of a given peptide.
Further disclosure by Gilon and coworkers (WO 95/33765) provided methods for producing building units required in the synthesis of backbone cyclized peptide analogs. Recently, The successful use of these methods to produce backbone cyclized peptide analogs having somatostatin activity was also disclosed (WO 98/04583 and WO 99/65508). Libraries of backbone cyclized analogs including IL-6 analogs are disclosed in international application WO 97/09344. In that disclosure, a selection method termed “Cycloscan”, based on conformationally constrained backbone cyclic peptide libraries that allows rapid detection of active analogs derived from a given sequence is described.
Nowhere in the background art, are backbone cyclized peptide analogs shown to possess IL-6 inhibitory activity.